UV/Vis και FTIR μελέτη της δέσμευσης μεθανικού και αιθανικού ανιόντος στην κυτοχρωμική οξείδαση ba3 από το Τhermus thermophilus

Abstract

The ba3-cytochrome oxidase, belongs to the superfamily of heme-copper oxidases that has been isolated from the bacterium Thermus thermophilus. It is an enzyme of approximately 85 kDa, which is composed of 764 residues, one heme b, one heme a3, three atoms of copper, proton transport channels, electrons and oxygen, a set of 15 transmembrane helices and a small periplasmic domain. It is the most important enzyme of the respiratory membrane protein complex of aerobic microorganisms, since it has the ability to catalyze the four electron reduction of molecular oxygen to water, at the same time it being oxidized. The reduction of oxygen is coupled to produce a kinetic power proton leading to the synthesis of ATP. The aim of this spectroscopic study is the observation of the binding of methanic and ethanoic anion to the binuclear center of cytochrome ba3. Methane anion binding to other cytochrome oxidases is still controversial as far as the way, but also the oxidation state of the enzyme. Through the use of Ultraviolet-visible spectroscopy (UV/Vis) and infrared (FTIR) we are able to observe the possible joining of ligands (substituents), either as a commitment to the active site, or to a secondary area (cavity) capture patient. Such kinds of cavities are essential to the catalytic activity of the enzyme, as they control thermodynamic and kinetic progress of the reaction. The two substituents used in this study may serve as detectors of the electrostatic and dynamics interactions present in the active site of the protein. The spectrophotometric methods in the visible-ultraviolet and infrared, are respectively the most widely used and reliable techniques for qualitative and quantitative analysis, capable to give selective responses to investigate the structure of matter at molecular and individual level. The results of the study through Ultraviolet-visible (UV/Vis) spectroscopy, show an interaction of both methane and ethane anion with the binuclear site of the enzyme. The indications are the same for both reduced and oxidized form of the enzyme and are attributed to weak binding of ligands to an inner-cavity area of the protein. The FTIR results, by detecting characteristic vibrations, are compatible with the deprotonated form of the two substituents which are subjected to interactions of electrostatic nature with amino acid residues located around this cavity. The two ligands, via the observed vibrational shifts in the infrared spectrum, operate as detectors of these electrostatic interactions and give important information about the immediate environment of the active binuclear center of the enzyme.